key: cord-0770829-wuqsfv5b
authors: Qiu, Hongyu; Yuan, Xin-Yong; Cabral, Teresa; Manguiat, Kathy; Robinson, Alyssia; Wood, Heidi; Grant, Chris; McQueen, Peter; Westmacott, Garrett; Beniac, Daniel R.; Lin, Lisa; Carpenter, Michael; Kobasa, Darwyn; Gräfenhan, Tom
title: Development and characterization of SARS-CoV-2 variant-neutralizing monoclonal antibodies
date: 2021-11-08
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2021.105206
sha: 3ac1c85c002f70652261e39110d43a72a48dc720
doc_id: 770829
cord_uid: wuqsfv5b

Vaccination and administration of monoclonal antibody cocktails are effective tools to control the progression of infectious diseases and to terminate pandemics such as COVID-19. However, the emergence of SARS-CoV-2 mutants with enhanced transmissibility and altered antigenicity requires broad-spectrum therapies. Here we developed a panel of SARS-CoV-2 specific mouse monoclonal antibodies (mAbs), and characterized them based on ELISA, Western immunoblot, isotyping, and virus neutralization. Six neutralizing mAbs that exhibited high-affinity binding to SARS-CoV-2 spike protein were identified, and their amino acid sequences were determined by mass spectrometry. Functional assays confirmed that three mAbs, F461G11, F461G15, and F461G16 neutralized four variants of concern (VOC): B.1.1.7 (alpha), B.1.351 (beta), P.1 (gamma) and B.1.617.2 (delta) These mAbs are promising candidates for COVID-19 therapy, and understanding their interactions with virus spike protein should support further vaccine and antibody development.

Unlike any other outbreaks of an infectious disease in recent history, the coronavirus 2019 pandemic has changed public health, the global economy, and people's lifestyle. It has resulted in over 192 million confirmed cases and more than 4.1 million deaths worldwide as of 2021 July 23 (https://covid19.who.int/), since its outbreak was recognized in December 2019. It is still not clear when this pandemic will be contained or controlled (Scudellari, 2020) . Although SARS-CoV-2 is not as lethal as SARS-CoV and MERS, it is much more contagious, transmitting efficiently through direct contact, respiratory droplets and aerosols through speaking, coughing and sneezing (Wiersinga et al., 2020; Editor, 2020) . Keeping a social distance, washing hands, or using hand sanitizer, and wearing masks in enclosed spaces have proven to be effective measures to slow down transmission. Failure to follow these measures can result in a surge of infections followed by exhaustion of medical resources such as hospital beds, ICUs, and ventilators, and significantly increased deaths (Ergonul et al., 2021) , a scenario which many regions have experienced during this pandemic (Onder et al., 2020; Meyerowitz et al., 2020) .

Central to controlling the pandemic is vaccination and a number of effective vaccines, developed against the Wuhan wild-type strain, are being administered on a global level (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/covid-19-vaccines). At this point, all current vaccines target the virus spike protein with the intent of developing neutralizing antibodies against this protein in order to block virus entry into cells (Dong et al., 2020; Krammer et al., 2020) . In J o u r n a l P r e -p r o o f addition, vaccines that elicite strong cellular response were also explored (Dong et al., 2020; Krammer et al., 2020) .

As an additional therapeutic measure, neutralizing antibodies, alone or in cocktails, are being assessed.

Three SARS-CoV-2-neutralizing monoclonal antibody (mAb) products: REGEN-COV (casirivimab plus imdevimab) (Hansen et Although vaccination and antibody cocktail prophylaxis/therapy seems promising, the emergence of SARS-CoV-2 variants with increased transmissibility, virulence, and antibody-resistance has raised concerns on the success of halting the pandemic (Zhou et J o u r n a l P r e -p r o o f

VeroE6 cells were infected at low moi (<0.01) with SARS-CoV-2 (hCoV-19/Canada/ON_ON-VIDO-01-2/2020, GISAID accession# EPI_ISL_425177) in neat DMEM. After 3 days, supernatant was pooled and cell debris pelleted by centrifugation at 1500xg for 15 min. Virus was pelleted by centrifugation at 28,000 rpm for 1hr, and washed with PBS. Titer prior to inactivation was determined by standard plaque assay.

The virus was inactivated by treatment with 0.1% formaldehyde solution for 3 days at 4 o C. Inactivation was confirmed by passage of 10-fold serial dilutions of the inactivated virus stock on VeroE6 cells with repassage of the culture supernatants onto fresh VeroE6 cell cultures for another 3 days to confirm lack of cytopathic effect that would be due to virus replication.

The coding sequence of the SARS-CoV-2 spike ectodomain amino acids 1-1215 (NCBI Accession # MN908947), was modified (Pallesen et al., 2017) , and placed in frame with a Thrombin cleavage site, T4 foldon trimerization motif (Tao et al., 1997) , a Strep tag II (Schmidt and Skerra, 2007 ) and a FLAG tag (Hopp et al., 1988) . The sequence was codon optimized for human cell expression, and cloned into Female BALB/c mice (5-6 weeks old) were immunized subcutaneously with 2x10 6 pfu formalininactivated SARS-CoV-2 virus in PBS with an equal volume of Emulsigen-D adjuvant (MVP adjuvants.

Omaha, NE, USA), and boosted at days 21, 35, 49, and 63, respectively. The mice were further boosted subcutaneously with 3x10 6 pfu formalin-inactivated SARS-CoV-2 virus three days before cell fusion.

J o u r n a l P r e -p r o o f

Cell fusion and mAb generation were conducted as described before (Berry et al., 2004) . Enzyme-linked immunosorbent assay (ELISA), Western immunoblot, and antibody isotyping also followed protocols described previously (Berry et al., 2004) .

Antibody digestion procedures were developed from protocols described in IgBLAST (https://www.ncbi.nlm.nih.gov/igblast/index.cgi) was used for germline gene alignment and allele determination based on protein sequences. Amino acid sequences were also back-translated into DNA sequences via EMBOSS Backtranseq (https://www.ebi.ac.uk/Tools/st/emboss_backtranseq) for further analysis based on nucleic acid sequences using IMGT/V-QUEST ( http://www.imgt.org/IMGT_vquest/analysis) and IgBLAST.

Purified mAbs were tested for antigen binding titers to SARS-CoV-2 rSP by ELISA described previously (Berry et al., 2004) . Endpoint titers of each sample were determined as the lowest concentrations of the wells at which the optical density (OD) was threefold higher than the negative control.

J o u r n a l P r e -p r o o f Measurement of the affinity of the mAbs for rSP was performed as described using a Biacore 2000 instrument (Biacore, Uppsala, Sweden) (Supplementary Materials and Methods). BIAevaluation 3.2 software was used to measure and plot the kon and koff values directly, which were used to calculate the affinity (KD).

The SARS-CoV-2 Surrogate Virus Neutralization Test Kit (GenScript, Piscataway, NJ, USA) was used to detect neutralizing antibodies against SARS-CoV-2 that block the interaction between the receptor-binding domain (RBD) of the viral spike glycoprotein with the ACE2 cell surface receptor.

The hybridoma culture supernatants and six purified neutralizing mAbs were tested at the same antibody concentration (1mg/ml). The SARS-CoV-2 PRNT was adapted from a previously described method for 

Four mice were immunized and boosted with formalin-inactivated SARS-CoV-2, and the spleens were used for fusion and hybridoma selection.

A panel of forty-four clones were detected based on ELISA screening against purified inactivated SARS-CoV-2, recombinant spike protein (rSP), recombinant nucleoprotein (rNP), and in parallel with negative screening with BSA. Forty-one clones that reacted with rSP or rNP, but not BSA, ( Table 1) were selected J o u r n a l P r e -p r o o f for antigen specificity confirmation by western immunoblot using non-reducing SDS-PAGE condition.

Twenty-eight mAbs reacted specifically with SARS-COV-2 spike protein, and thirteen mAbs reacted specifically with nucleoprotein.

Isotype analysis found that most clones are IgG/k type mAbs, while F461G5 and 461G12 contain IgG/λ. F457G4, F461G14, and F461G17 initially contained both IgG and IgM. After two rounds of subcloning, only IgG was selected from F457G4 and F461G14. F461G17 and F458G1 stopped producing antibodies during subcloning and were excluded in the following analyses (Table 1) .

Unpurified hybridoma culture supernatants were used for the SARS-CoV-2 surrogate virus neutralization test (sVNT), and eight clones (F457G11, F459G1, F461G8, F461G11, F461G14, F461G15, F461G16, and F461G17) showed potential virus neutralization capability (≥ 20% inhibition rate of the positive control). Of those, six mAbs were purified and further identified based on plaque reduction neutralization test (PRNT) using a SARS-CoV-2 isolate (Canada/ON_ON-VIDO-01-2/2020, EPI_ISL_42517) ( Table   1 ). All nucleoprotein-specific mAbs didn't show neutralization capacity, and their characteristics is under investigation and will be reported in a future study.

Based on the preliminary live virus neutralization results, a lead panel of six spike protein-specific mAbs (F459G1, F461G8, F461G11, F461G14, F461G15, and F461G16) were selected for subcloning and production on a large scale for testing antibody sequence, antigen specificity, antigen affinity, surrogate virus neutralization test, and neutralization of multiple SARS-CoV-2 variants.

Anti-SARS-CoV-2 antibody amino acid sequences were determined using mass spectrometry based denovo sequencing of purified monoclonal antibody digests. Template sequences for each antibody were exported from PEAKS AB and considered to be the primary amino acid sequence. All six antibody sequences were generated from the results of two replicate digestion sets except for F461G14, which was from the results of a single digestion set. We were able to obtain 100% sequence coverage for all antibodies. Each sequence was evaluated manually for annotation of CDR and amino acid confidence. All the amino acids in F461G1, F461G11, F461G16 heavy and light chain sequences were identified with J o u r n a l P r e -p r o o f >95% confidence. All amino acids in F461G14 and F461G15 were identified with >95% confidence except for two amino acids in the CDR3 that were identified with >85% confidence. F461G8 had three unidentified amino acids, two with >85% confidence and one with >95% confidence of the six CDR3 amino acids.

The sequences of the six neutralization mAbs used three heavy-chain-variable genes, with the VH5-9-1*01 gene most frequently used by four mAbs (F461G11, F461G14, F461G15 and F461G16) ( Table 2) .

While the light-chain of the six mAbs belong to two gene families, using three light-chain-variable genes, with the VK4-80*01 used by three mAbs. The sequence identity to germline varied from 75.5% to 96.9% for heavy chains and from 80.0% to 96.8% for light chains, respectively. The broad range of sequence identity to the germline genes is consistent with repeating exposure to the antigen. The same heavy and light chain genes were used by F461G14, F461G15, and F461G16, suggesting they were derived from related hybridoma clones.

All six mAbs reacted specifically with the SARS-CoV-2 rSP, but not BSA (negative control) in Western immunoblots. These antibodies reacted with spike protein only in non-reducing conditions, but not in reducing SDS-PAGE (with DTT). This indicated that these mAbs targeted conformational epitopes maintained by disulfide bonds (Supplementary Figure 1) .

Both ELISA and Biacore were used to determine the binding affinity of selected mAbs with rSP.

All six mAbs showed strong binding with spike proteins. Two mAbs, F461G8 and F461G11, had endpoint titers at 156 ng/mL. While the other four mAbs, F459G1, F461G14, F461G15, and F461G16 showed stronger binding with the endpoints at 19.5 ng/mL, 4.9 ng/mL, 4.9 ng/mL, and 3.1 ng/mL, respectively.

Biacore analysis demonstrated a similar trend of rSP-antibody interaction as indicated by endpoint ELISA. Both F461G8 and F461G11 showed binding affinity at the nanomolar level, while the other four mAbs had a stronger affinity to the S protein, with the KD below 1nM (Table 3) .

J o u r n a l P r e -p r o o f

Both sVNT and PRNT tests were used for effector function analysis. sVNT is a fast and easy way to screen the antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. In contrast, the PRNT is a more accurate assay by using the clinical isolated virulent virus. However, it can only be done in a biosafety level 3 (BSL3) lab and is labor-intensive.

The preliminary tests using the hybridoma culture supernatants demonstrated the neutralizing capacity of the six lead mAbs. Purified mAbs were further tested to determine the effects of selected clones. As shown in Table 3 , all six mAbs demonstrated strong neutralization capacity using the sVNT, with 96% or more blocking of the RBD binding with ACE2, compared to the positive control. Because of the nature of this test, the results also suggest that the epitope binding domains of the six neutralizing mAbs are located in the receptor-binding domain (RBD).

The six mAbs that showed high blocking values were further characterized in the PRNT against the SARS-CoV-2 clinical isolate used for hybridoma development. All mAbs demonstrated strong SARS-CoV-2 PRNT90 titers ranging from 80 to 320. In summary, all selected six mAbs demonstrate substantial neutralization potency against the SARS-CoV-2 virus.

As the pandemic has progressed, several SARS-CoV-2 variants have emerged and spread in different . Therefore, we also tested the neutralization activity of these lead mAbs against the variants of concern (VOCs) present in North America (https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variantinfo.html#Concern) and other continents.

The neutralization potency of the candidate mAbs to the four VOCs are ascribed into three categories: 1, significantly reduced compared to most of the variants (F461G8 and F461G14, more than a thirty twofold reduction of the neutralization activity to two (F461G8) to three (F461G14) VOCs, and eight-fold reduction to another VOC (F461G8)), 2, moderately reduced (F459G1, four to sixteen-fold reduction of the neutralization activity to three VOCs), and 3, unchanged or improved neutralization (F461G11, F461G15, and F461G16, up to two-fold decrease and eight-fold increase of the neutralization activity to the VOCs) (Figure 1 ).

Both F461G8 and F461G14 showed a substantial reduction of neutralization potency to most of the tested variants. Specifically, the neutralization potency of F461G8 to variants P.1 and B.1.351, decreased up to thirty-two fold, and the potency to variant B.1.1.7 and B.1.617.2 decreased two-fold and eight-fold (by PRNT50), respectively. F461G14 showed neutralizing activity at more than thirty-two-to sixty-four--fold reduction to variants P.1, B.1.1.7, In summary, F461G11, F461G15, and F461G16 demonstrated strong neutralizing activity to a broad spectrum of clinical isolates, including the current four variants of concern that are prevalent in various geographical regions.

The COVID-19 pandemic has heavily hit public health and the global economy during the last one and a All forty-four mAbs generated in this study were pre-screened using sVNA, and thirty-three were further tested by PRNT. Except for F457G11 and F461G17, all the pre-screening results were consistent with the neutralization of the isolate used for mouse immunization ( Table 1) All the leading six neutralizing antibodies in this study were pre-screened using the surrogate virus neutralization assay, based on their binding to the RBD of SARS-CoV-2 spike protein (Tan et al., 2020) .

However, it is essential to point out that some antibodies can recognize other domains within the spike protein, such as the NTD of S1, or fusion loop of S2, which may also interfere with the interaction between the RBD and ACE2, or membrane fusion, and inhibit virus entry into the host cell (Jiang et al., 2020; Chi et al., 2020). Therefore, assessment of the other mAbs based on virus neutralization test is needed to fully characterize all spike protein-specific antibodies.

Overall, amino acid sequencing of antibodies using a combination of shotgun proteomic techniques and PEAKS AB de-novo sequencing was able to provide high quality sequence information. Most amino acid sequences were determined with >95% confidence while only F461G8 had three unidentified amino acids in the CDR3. CDR3 is a region of high variability so it is natural to assume that challenges would be observed in sequencing this antibody region. Sequencing in this region will largely be dependent on the quality of de-novo determined sequences and the amount of variation in this region between homologous sequences. In most cases, we were able to improve sequence information in this region by analysing multiple replicate digests of the same antibody. Ongoing work will continue to further improve the accuracy of amino acid sequences of the antibodies, for example by using different digestion enzymes and/or peptide fragmentation methods.

Four neutralizing antibodies (F461G11, F461G14, F461G15, and F461G16) share the same germline heavy chain V gene (IGHV5-9-1*1) and three of them (F461G14, F461G15, and F461G16) share the same germline light chain V gene (IGKV4-80*01) as well. In contrast, the light chain of F461G11 was suggested from IGKV6-20*01 (80.0% identity). Further analysis found that it is 79.6% identical with IGKV4-80*01, indicating that F461G11 was also derived from hybridoma clone related the other three mAbs. Given the similarity of F461G11, F461G15, and F461G16 in sequences and neutralization potency on the VOCs, it is speculated that they share similar antigen epitopes. It is also intriguing that F461G14, although has similar germline heavy and light chain V genes as the other three, can not neutralize three VOCs (alpha, beta, and gamma variants). Antibody binning and epitope mapping will be conducted to answer these questions.

In summary, we identified three SARS-CoV-2 spike protein-specific murine mAbs that demonstrate broad-spectrum neutralization of the variants of concern. We are working on epitope binning and mapping, and the mAb-spike protein structure determination. Understanding the interactions between these mAbs and the spike protein is expected to shed light on developing a new generation vaccine against a broad spectrum of SARS-CoV-2 variants. In addition, a humanized version of these mAbs, after thorough characterization of the efficacy and safety (EMA, 2016; WHO, 2016; FDA, 2021), might also provide more therapy options for COVID-19. biotin. Samples were further purified by Superdex Increase size exclusion chromatography (Cytiva) .

Protein concentrations were determined by Qubit analysis (Thermo Fisher) and adjusted to 0.5 -1.0 mg/ml in PBS prior to being snap-frozen in liquid nitrogen. Protein purity, subunit trimerization and the absence of aggregates was confirmed by SDS PAGE and Superdex Increase size exclusion chromatography.

Recombinant NP was expressed and purified using a similar regimen. A codon optimized NP sequence was placed downstream of a BM40 secretion signal and upstream of a thrombin recognition sequence, a streptag II motif and a FLAG tag domain. Expression in 293 Expi cells was for 4 days prior to harvest and purification as described above for recombinant spike protein. Tables   Table 1 Identification 

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All solutions were purchased from

mixture of 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide hydrochloride:N-hydroxysuccinimide (EDC:NHS). were diluted in sodium acetate (pH 5.5), and 20 µL of this solution was used to coated on the activated chip. The chip was then blocked by the addition of 35 µL of ethanolamine-HCl, followed by a wash with 35 µL of 10 mM glycine-HCl (pH 1.5). The anti-spike mAbs were diluted in HBS-P buffer to final concentrations ranging from 20µg/mL to 100µg/mL, and 40 µL of each dilution (five dilutions in total for each mAb) was applied in turn to the rSP-coated flow cell. The flow cell surface was

BIAevaluation 3.2 software was used to measure and plot the kon and koff values directly, which were then used to calculate the affinity (KD). The best fit binding curves were generated assuming a one-to-one binding interaction

Antibodies were diluted 1:10 in DMEM supplemented with 2% FBS and 1X penicillin. In a 96-well plate, the antibodies were further diluted 2-fold from 1:10 to 1:1280 in DMEM supplemented with 2% FBS and 1X penicillin in a volume of 150 μL. One hundred fifty microlitres of SARS-CoV-2 diluted at 100 PFU/100 μL was added to each well

No neutralization, 50% neutralization, and 90% neutralization controls were prepared by diluting SARS-CoV-2 at 50 PFU/100 μL, 25 PFU/100 μL, and 5 PFU/100 μL, respectively. DMEM supplemented with 2% FBS and 1X penicillin was used as a no virus control. After 1 hour of incubation

This work was supported by National Microbiology Laboratory, Public Health Agency of Canada (Project number: BI-2020-01-I-000000-SCV-081). We are grateful to NML's Veterinary Technical Services team for the excellent and timely support with the immunization of mice and other animal procedures. We also thank Estela Ochoa for hybridoma development and characterization, and Stuart McCorrister in the Mass Spectrometry & Proteomics Core Services unit for the high-quality nanoflow-LC/MS/MS data acquisition.